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Target-specific multiple sequencing primer pool for microbial typing and sequencing applications in DNA-sequencing technologies

a technology of dnasequencing technology and primer pool, which is applied in the direction of microorganism testing/measurement, sugar derivatives, biochemistry apparatus and processes, etc., can solve the problems of difficult or impossible base calling, and achieve good results and improve sequence data quality

Inactive Publication Date: 2005-09-15
GHARIZADEH BABACK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0025] Hereby, by providing a mixed set of oligonucleotide primers to the same sample only the primers having a complementary nucleotide sequence in the sample will hybridize, and thus only the type(s) related to these primers will result in sequence signals. Accordingly, this method is highly advantageous when analyzing a sample comprising several types of one variable / target site, such as a multiple-infected sample.
[0038] The new method was also used for faster and more specific typing of bacteria. Bacterial DNA was amplified with PCR utilizing general primers binding to conservative regions on the 16S rRNA gene. The bacterial amplicons used in this study could be grouped in two categories, one group with 19 bases sequence similarity downstream of the PCR primer and the second group with 31 bases sequence similarity. Two sequence-group specific sequencing primers were designed and added together to the amplified bacterial DNA. Fast and correct typing of bacteria from both groups was possible by the Pyrosequencing technology. The method was applicable to Sanger DNA sequencing as well. The new method is especially useful in combination with the Pyrosequencing technology (this method for the moment is limited to sequencing about 50-100 bases depending on the template) as the method of the present invention is not dependent on starting the sequencing in a conserved region of the DNA. Thereby less of the number of bases that can be amplified using the Pyroseuqnecing technology are “wasted” on sequencing conserved regions and more of the number of bases that can be sequenced are actually used for sequencing of the variable regions.

Problems solved by technology

The amplicon might also contain unspecific amplification, giving rise to sequence signals making base calling difficult or impossible.

Method used

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  • Target-specific multiple sequencing primer pool for microbial typing and sequencing applications in DNA-sequencing technologies
  • Target-specific multiple sequencing primer pool for microbial typing and sequencing applications in DNA-sequencing technologies
  • Target-specific multiple sequencing primer pool for microbial typing and sequencing applications in DNA-sequencing technologies

Examples

Experimental program
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Effect test

example 1

Materials for HPV Typing

[0085] The primers for example 1 are designed for the HPV types that can be amplified with GP5+ / 6+ or MY09 / 11 primers sets. Each sequencing primer is designed to be specific for one designated HPV genotype. All the primers were checked by BLAST search for specificity and the resulting sequences were specific for the type designed.

[0086] The specific HPV-primers designed for detection of high-risk HPV by the multiple sequencing primer strategy of the invention were:

[0087] High-risk HPV genotypes

HPV-165′-GCTGCCATATCTACTTCAGAHPV-185′-GCTTCTACACAGTCTCCTGTHPV-315′-GTG CTG CAA TTG CAA ACA GTHPV-335′-ACACAAGTAACTAGTGACAGHPV-455′-TATGTGCCTCTACACAAAAT

[0088] Low-risk HPV genotypes

HPV-65′-GTG CAT CCG TAA CTA CAT CTTHPV-115′-GTG CAT CTG TGT CTA AAT CTG

[0089] The GP5+ / 6+ amplification primer set sequence is:

GP5+5′-TTT GTT ACT GTG GTA GAT ACT AC 3′Biotin-GP6+5′-GAA AAA TAA ACT GTA AAT CAT ATT C 3′

[0090] The MY09 / 11 amplification primer set sequence is:

Biotin-MY0...

example 2

Typing of HPV in Clinical Samples and Simulated Mixed-infections by Applying Multiple Sequencing Primers in Pyrosequencing Technology

[0091] Some types of human papillomaviruses (HPV) are broadly recognized on a global scale as causative agents for cervical cancer. They are also related to other cancer types. For genotyping of HPVs we introduced the Pyrosequencing technology for specific HPV-typing (Gharizadeh et al., 2001). In general, not more than 25 bases are needed for specific genotyping. Multiple infections and unspecific amplification have been a problem for typing as double / multiple infections or unspecific amplification products present in one specimen give rise to sequence signals from all available types / products in the specimen. This is mainly because all HPV types in a sample are amplified with PCR utilizing the general GP5+ / 6+ primer set and then sequenced utilizing the GP5+ primer as extension / sequencing primer. This primer hybridizes to all present types, or unspeci...

example 3

Typing of HPV in Mixed-infections by Dideoxi DNA-sequencing (Sanger)

[0097] The general approach of example 2 was repeated, and Sanger sequencing was used as the DNA-sequencing method, instead of Pyrsosequencing (all the figures are not shown). FIG. 11a shows HPV-16 / 72 / 6-mixture sequenced with a general primer as extension primer. FIG. 11b shows HPV-16 / 72 / 6-mixture, sequenced by the primer set of the invention, which comprises a mix of 4 primers (HPV-16, 18, 33 and 45) (Gharizadeh et al., 2003). The results show clearly that with the dideoxy sequencing, typing with the primer set of the invention renders sequencing results that are clear to interpret than by using a general primer (impossible). An ABI prism DNA analyzer 3700 (Applied biosystems) was used for the DNA-sequencing with Big dye terminator kit according the manufacturer's manual. Same results were achieved on amplicons derived by MY09 / 11 using a-seven-multiple sequencing primers (unpublished data).

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Abstract

The invention relates to a method of typing a sample of nucleic acid molecules in DNA sequencing technologies, whereby the molecules are suspected to comprise at least DNA from one type or species, e.g. microorganisms or viruses, with each species or type or target DNA having different nucleotide patterns. By adding a mixed set of at least two oligonucleotide primers, whereby each primer is designed for being specific for one type or species or target DNA, thereby allowing a primer or primers to hybridize in or close to the type-specific or target regions; determining the types or targets sequence by extending the hybridized primer or primers in a sequencing reaction. Hereby, the typing is simplified by using a set of type-specific primers and sequence pattern recognition approach. The method of invention is suitable for samples containing a plurality of types or species, multiple pathogenic infections or variants, and amplicons with unspecific amplification products. Furthermore, the invention relates to a kit for use in the method.

Description

[0001] Note: This patent application is related to the provisional patent application with the application No. 60 / 399,357 filing date Jul. 7, 2002.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] The invention is not federally sponsored. Abstract [0003] The invention relates to a method of typing a sample of nucleic acid molecules, whereby the molecules are suspected to comprise at least one type / species / target DNA of a type-specific / target region chosen from a known set of types of the type-specific / target region, each species / type / target having different nucleotide patterns, comprising the steps of: providing the sample of nucleic acid molecules; providing a mixed set of at least two oligonucleotide primers, whereby each primer is designed for being specific for one type / species / target DNA chosen from the known set of types of the nucleic acid sample, thereby allowing a primer, which is specific for a type that is present in the sample, to hybridize in or clo...

Claims

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Application Information

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IPC IPC(8): C07H21/04C12Q1/68C12Q1/70
CPCC12Q2600/16C12Q1/708
Inventor GHARIZADEH, BABACK
Owner GHARIZADEH BABACK
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